Abstract The chemokine receptor CXCR4 is a critical regulator of cell migration and serves as a coreceptor for HIV-1. The chemokine stromal cell derived factor-1, also known as CXCL12, binds to CXCR4 and exerts its biologic functions partly through the small guanosine triphosphate hydrolase (GTPase) Rac1 (ras-related C3 botulinum toxin substrate 1). We show in different cell types, including CD34+ hematopoietic stem and progenitor cells, that inhibition of Rac1 causes a reversible conformational change in CXCR4, but not in the related receptors CXCR7 or CCR5. Biochemical experiments showed that Rac1 associates with CXCR4. The conformational change of CXCR4 on Rac1 inhibition blocked receptor internalization and impaired CXCL12-induced Gαi protein activation. Importantly, we found that the conformation adopted by CXCR4 after Rac1 inhibition prevents HIV-1 infection of both the U87-CD4-CXCR4 cell line and of primary peripheral blood mononuclear cells. In conclusion, our data show that Rac1 activity is required to maintain CXCR4 in the responsive conformation that allows receptor signaling and facilitates HIV-1 infection; this implies that Rac1 positively regulates CXCR4 function and identifies the Rac1-CXCR4 axis as a new target for preventing HIV-1 infection.